Quantitative determination of serum calcium



United States Patent 3,457,045 QUANTITATIVE DETERMINATION OF SERUMCALCIUM Jose M. Fraguada, Dover, and Robert W. Le Beau, Bernardsville,N.J., assignors to Warner-Lambert Pharmaceutical Company, Morris Plains,NJ., a corporation of Delaware No Drawing. Filed Apr. 25, 1966, Ser. No.544,837 Int. Cl. G01n 33/16, 31/02 U.S. Cl. 23-230 6 Claims ABSTRACT OFTHE DISCLOSURE Composition and process for quantitatively determiningthe amount of calcium ion in blood serum. A measured quantity of bloodserum is treated with an aqueous solution of measured quantities ofsodium rhodizonate and sulfosalicylic acid; proteins and interferingchromogens from the blood serum are precipitated out by thesulfosalicylic acid. The clear solution remaining after the removal ofthe precipitate contains a colored calcium rhodizonate formed by thereaction of the sodium rhodizonate with substantially all of the calciumions present in the blood serum. The reaction is quantitative and theintensity of the color, as measured at a wave length of 550 ml, iscompared against known standard solutions of calcium rhodizonate. Theaddition of the sulfosalicylic acid and sodium rhodizonate solution tothe blood serum being tested can be performed consecutively, but forconvenience, simultaneous addition of 'both reagents is preferred.

The present invention relates to a composition and process fordetermining quantitatively the amount of calcium ion in blood serum, andmore particularly relates to the use of sodium rhodizonate as adiagnostic agent for the quantitative determination of serum calcium.

Sodium rhodizonate is known to be useful as a qualitative agent for thedetermination of the presence of alkaline earth metals such as barium,strontium and calcium in unknown chemical samples.

It has been discovered that sodium rhodizonate (i.e., the 'disodium saltof rhodizonic acid) reacts specifically and quantitatively with serumcalcium to produce a highly colored compound, the intensity of color ofwhich depends on the concentration of calcimum ion present in the bloodserum being tested, i.e., on the amount of calcium rhodizonate formed.The resulting colored solution can then be compared against knownstandard solutions of calcium rhodizonate at a wave length of 550 m on aspectrophotometer or a colorimeter to determine the quantity of calciumions present in the blood serum sample.

The instant procedure is specific for calcium in blood serum since thereare no significant quantities of any other alkaline earth metal ionssuch as barium or strontium ions in blood serum.

The determination of the amount of calcium ion in blood serum is, ofcourse, an important diagnostic tool in the determination of certainphysiological disorders in the animal body such as those causing anelectrolyte imbalance, impairment of calcium utilization, etc.

The process of the invention is carried out by reacting 3,457,045Patented July 22, 1969 a given quantity of blood serum with from about0.0204 to about 0.0276, preferably about 0.0216 to about 0.0264 part ofsodium rhodizonate and from about 2.55 to about 3.45, preferably 2.70 toabout 3.30 parts of sulfosalicylic acid, preferably as the dihydrate,made up into an aqueous solution with distilled or deionized water. Theabove parts are parts by weight per part by weight of blood serum..

The above aqueous solution contains from about 0.00136 to about 0.00184part by weight, preferably about 0.00144 to about 0.00176 part by weightof sodium rhodizonate and from about 0.17 to about 0.23 part by weight,preferably about 0.18 to about 0.22 part by weight of sulfosalicylicacid, per part by Weight of Water. The sulfosalicylic acid dihydrate isemployed to precipitate proteins and interfering chromogens from theblood serum. For convenience, the sulfosayicylie acid dihydrate isemployed simultaneously with the sodium rhodizonate in the samesolution, although, if desired, all of the sulfosalicylic acid dihydratecan be dissolved in about half of the above quantity of water and mixedwith the blood serum, followed by the addition of all of the sodiumrhodizonate dissolved in the other half of the water.

The above solution of sodium rhodizonate and sulfosalicylic acid, or theseparate solutions of each, are mixed with the sample of blood serum andthe precipitated protein and chromogens are removed, e.g., bycentrifugation or filtration. The resulting clear solution is then addedto from about 3 to about 13 parts, preferably about 7 to about 9 partsby volume of a solution of from about 40% to about preferably about 60%glycerol in distilled or deionized water. The resulting solution ismixed well, and from about 1.75 to about 3.0 parts by volume, preferablyabout 2 parts 'by volume, based on the above clear solution obtainedprior to addition of the glycerol solution, of a 1.0 N solution ofcarbonatefree alkali metal hydroxide, preferably sodium hydroxide, indistilled o1- deionized water is carefully added to the above solutionas an upper layer. One or more known standards (i.e. solutionscontaining known quantities of calcium ion) are similarly preparedaccording to the above process together with a bland (i.e., instead ofblood serum or an equal quantity of a solution containing a knownquantity of calcium ion, an equal quantity of distilled or deionizedwater is employed in the above process to form the blank). Whatevervalues are chosen within the above ranges, they must, of course, beidentical for the standards and the blank. The above solutions are thenmixed simultaneously and the colors allowed to develop undisturbed forfive minutes. At the end of five minutes the absorbances of the unknownand the standards are read against the blank set at zero CD. at 550 mpwave length.

The quantity of calcium present in the unknown blood serum sample canreadily be determined by simple standard calculations as shownhereinafter.

The invention will be better understood from a consideration of thefollowing example which is given for illustration purposes only and isnot meant to limit the invention.

EXAMPLE (a) Reagent A Into a one (1) liter volumetric flask place: 500mls.

of distilled or deionized water and 800 mg. of sodium rhodizonate.

MIX until the sodium rhodizonate is dissolved completely. Add 100.0grams of sulfosalicyclic acid.

Mix until acid is dissolved completely and make up to volume withdistilled or deionized water. (b) Reagent B 60% solution of glycerol isdistilled or deionized water. (c) Reagent C 1.0 normal solution ofsodium hydroxide in water, standardized, carbonate free.

Procedure (1) Into three 13 x 100 mm. test tubes place:

(a) 3.0 mls. of Reagent A in each tube.

(b) 0.2 ml. of serum in the first, 0.2 ml. of standard in the second and0.2 ml. of water (blank) in the third tube.

(2) Mix the contents of each tube well and centrifuge at 2,500 r.p.m.for 5.0 minutes.

(3) Into three 19 x 105 mm. test tubes place:

(a) 5.0 mls. of Reagent B in each tube.

(b) 1.0 ml. of supernatant liquid from the first 13 x 100 mm. tube inthe first 19 x 105 mm. tube, 1.0 ml. of supernatant liquid from thesecond 13 x 100 mm. tube in the second 19 x 105 mm. tube and 1.0 ml. ofsupernatant liquid from the third 13 x 100 mm. tube in the third 19 x105 mm. tube, and mix the contents of each tube well.

(c) Slant tubes, slowly and carefully overlay 2.0 mls. of

Reagent C in each tube.

Note: At this state, the interphase between the two solutions in thethree tubes should be colorless or slightly colored.

Notes:

(1) Absorbances should be read within a period of ten minutes.

(2) Do not shake tube with color developed. Color tends to fade fast ifshaken.

Calculation:

O.D. of Unknown X Concentration of Standard= Concentration of UnknownStandardization:

Process for the preparation of a 20 mg. percent Ca stock standard (1)Dry overnight pure calcium carbonate in an oven (300 C.).

(2.) Since the molecular weight of CaCo is 100, then 1 the equivalentweight of CaCo is /2. or 50.0.

Standardization: Plotting Curve. 5 mg. percent Ca Standard:

5/20=0.25 20=5 mls. stock and 15 mls. water 10 mg. percent Ca Standard:

10/20=0.5 x20: 10 mls. stock+10 mls. water 15 mg. percent Ca Standard:

15/20=0.75 20= 15 mls. stock+5 mls. water 20 mg. percent Ca Standard:Use straight stock standard.

Mix standards well and carry out the above procedure for calciumdetermination using the standards as unknowns. Plot optical densityagainst concentration in mg. percent on graph paper. The resultingCartesian graph will be a straight line with five reference points. Thisline obeys Beer-Lamberts law from the origin (zero 0D.) to 20 mg.percent. As an alternative procedure to that given, unknown serumsamples can be measured using a blank, and the concentration of calciumin the serum read directly from the above graph.

What is claimed is:

1. A process for determining the amount of calcium ion in blood serumcomprising the steps of:

(a) treating a measured quantity of blood serum with an aqueous solutioncontaining from about 0.00136 to about 0.00184 parts by weight of sodiumrhodizonate and from about 0.17 to about 0.23 part by weight ofsulfosalicylic acid per part by weight of water, in amount sufiicient toprovide from about 0.0204 to about 0.0276 part by weight of sodiumrhodizonate and from about 2.55 to about 3.45 parts by weight ofsulfosalicylic acid per part by weight of blood serum,

(b) separating a clear solution from the resulting mixture,

(c) mixing said clear solution with from about 3 to about 13 parts byvolume of about 40% to an about 75% solution of glycerol in water,

(d) mixing the resulting solution with aqueous alkali metal hydroxide,

(e) mixing the above solution thoroughly and allowing it to standundisturbed for at least five minutes,

(f) measuring the intensity of the color at a wave length of 550 m in aspectrophotometer or colorimeter, and

(g) determining from the measurement the amount of calcium ion presentin the blood serum; wherein in the above steps any water present isdistilled or deionized water.

2. A process according to claim 1 wherein from about 0.00144 to about0.00176 part by weight of sodium rhodizonate and from 0.18 to about 0.22part by weight of sulfosalicylic acid are employed per part by weight ofwater, in amount sutficient to provide from about 0.0216 to about 0.0264part by weight of sodium rhodizonate and from about 2.70 to about 3.30parts by weight of sulfosalicylic acid per part by weight of bloodserum, in step (a) and from about 7 to about 9 parts by volume of theaqueous solution of glycerol is employed in step (c).

3. A process according to claim 1 wherein the determination in step (g)is carried out by comparison with the color intensity of a knownstandard.

4. A process according to claim 1 wherein the sulfosalicylic acid instep (a) is in the form of a dihydrate.

5. A composition useful in the determination of the calcium ion contentof blood serum comprising an aqueous solution of from about 0.00136 toabout 0.00184 part by weight of sodium rhodizonate and from about 0.17to about 0.23 part by weight of sulfosalicylic acid per part by weightof water; wherein said water is distilled or deionized water.

6. A composition according to claim 5 wherein from about 0.00144 toabout 0.00176 part by weight of sodium rhodizonate and from about 0.18to about 0.22 part by weight of sulfosalicylic acid are present per partby weight 15 of water.

References Cited Feigl, F., et a1.: Chemical Abstracts, vol. 48, p. 9259(1954).

Halok, P. B., et a1.: Practical Physiological Chemistry,

Mustatin, I. S., et a1.: Chemical Abstracts, vol. 54, pp. 11855-56(1960).

Sendroy, 1., Jr.: Journal of Biological Chemistry, pp. 539-56 1944MORRIS O. WOLK, Primary Examiner ELLIOTT A. KATZ, Assistant ExaminerU.S. Cl. X.R. 23-253; 252408

